Abstract

Background

HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert
particles as they contain no replicative nucleic acid, although they do encapsidate
cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems,
VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their
entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this
system facilitate uptake and subsequent expression of encapsidated RNA in mammalian
cells - an unfavourable characteristic for a vaccine.

Methods

HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The
presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect
and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA.
Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates
tested for the presence of CAT protein by ELISA. Mice were inoculated with heated
and unheated VLPs using a DNA prime VLP boost regimen.

Results

HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP)
on their surfaces. The amount of encapsidated CAT RNA/μg Gag VLPs ranged between 0.1
to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with
VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing
reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated
with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT
VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these
immune responses but enhanced the Gag CD4 T cell responses by two-fold.

Conclusions

Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected
mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA
was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability
of protein to be expressed in some cell lines tested but did not affect the ability
of the VLPs to stimulate an immune response when inoculated into mice.